LC/MRM‐MS experiments were performed in quintuplicate, by reversed‐phase UHPLC on a 1260 Infinity LC system using a Zorbax Eclipse Plus C18 Rapid Resolution HD column (150 × 2.1 mm, 1.8 μM particles; Agilent Technologies; Palo Alto, CA, USA). The column was maintained at 50 °C and the autosampler was kept at 4 °C. Mobile phase compositions of 0.1% FA in water for solution A and 100% ACN in 0.1% FA for B at a flow rate of 0.4 mL/min were used. The gradient was 0:2.7, 2:9.9, 15:17.1, 22:26.1, 25:40.5, 27:81, 29:81, and 30:2.7 (time, %B). The LC system was interfaced to a 6495 triple quadrupole mass spectrometer (both from Agilent Technologies) via a standard‐flow ESI source—see Table S1, Supporting Information for the list of the general acquisition parameters. Specific peptide parameters, such as collision energy and retention time, had been previously optimized and were not changed.45
Zorbax eclipse plus c18 rapid resolution hd column
Manufactured by Agilent Technologies
Sourced in United States
The Zorbax Eclipse Plus C18 Rapid Resolution HD column is a high-performance liquid chromatography (HPLC) column designed for rapid and efficient separation of a wide range of compounds. The column features a porous silica-based stationary phase with a C18 bonded ligand, providing excellent chromatographic resolution and peak shape. The Rapid Resolution HD technology enables fast and high-resolution separations, making it suitable for a variety of analytical applications.
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Lab products found in correlation
6495 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Agilent 1290 infinity 2, by Agilent Technologies (1 mentions)
6550 ifunnel q tof mass spectrometer, by Agilent Technologies (1 mentions)
1290 infinity hplc, by Agilent Technologies (1 mentions)
1290 infinity uhplc system, by Agilent Technologies (1 mentions)
7 protocols using zorbax eclipse plus c18 rapid resolution hd column
1
Quantitative Peptide Analysis via LC/MRM-MS
2
Enzymatic Sialylation Kinetics and pH Optimization
3
HPLC-QToF Analysis of Aflatoxin B1
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For AFB1, a 1,260 Infinity HPLC system, coupled to a 6,545 Quadrupole-Time-of-Flight (QToF) spectrometer, was used for the analysis, in conjunction with a mass detector (Agilent, Waldbronn, Germany) and control software (Mass Hunter Workstation; version B.06.11). The analysis parameters and conditions were as described by Iriondo-DeHond et al.21 . In brief, samples were injected into a Zorbax Eclipse Plus C18 Rapid Resolution HD Column (2.1 × 50 mm, 1.8 μm, Agilent, Santa Clara, CA) with a 5 mm guard column. The temperature was set at 30 °C and the mobile phase was 5 mM ammonium formate + 0.1% formic acid and 5 mM ammonium formate + 0.1% formic acid in methanol. The gradient elution was chosen as indicated in the procedure. Finally, compounds were identified and quantified using the ‘Find by Formula’ algorithm.
Samples were directly centrifuged according to the protocol described by Joannis-Cassan et al.22 (link). Different methodologies were proposed for the extraction of AFB1: extraction with organic solvents (ethyl acetate or methanol) or the use of a solid phase extraction column (ISOLUTE Myco, Biotage, Sweden). Quantification was achieved by injecting standard solutions from 0.005 to 0.04 mg/L at different sample volumes (10 µL and 30 µL).
Samples were directly centrifuged according to the protocol described by Joannis-Cassan et al.22 (link). Different methodologies were proposed for the extraction of AFB1: extraction with organic solvents (ethyl acetate or methanol) or the use of a solid phase extraction column (ISOLUTE Myco, Biotage, Sweden). Quantification was achieved by injecting standard solutions from 0.005 to 0.04 mg/L at different sample volumes (10 µL and 30 µL).
4
Quantification of Cantharidin in Beetles
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Chromatographic separations were performed on a Zorbax Eclipse Plus C18 Rapid Resolution HD column, 2.1 × 100 mm, 1.8 µm (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was performed by mixing of two mobile phase components (A and B) in an isocratic mode in the ratio 70:30. Mobile phase component A was deionized water and component B was methanol, both containing 0.1% formic acid. Sample volume 10 µL was injected into the column with a mobile phase flow rate 0.5 mL/min and temperature 45 °C. MS/MS analysis was carried out using ESI in positive ionization mode and the triple-quadrupole mass spectrometer operated in multiple reactions monitoring (MRM) mode in following conditions: source temperature 150 °C, desolvation temperature 500 °C, the capillary voltage 1.5 kV, the cone gas flow 20 L/h, and the desolvation gas flow 850 L/h. For measuring the presence of cantharidin, the transitions 197.0 > 94.99 and 197.0 > 123.0 were monitored. The quantification was done using matrix-matched calibration. The concentration of cantharidin was expressed to the dry weight of beetles.
5
Quantitative Analysis of Mycotoxins via LC-MS/MS
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Chromatographic separation was performed on a Zorbax Eclipse Plus C18 Rapid Resolution HD column (2.1 × 100 mm, 1.8 μm; Agilent). Aflatoxin separation was performed using mixture A (deionized water) and B (methanol) in a 60:40 ratio isocratic condition. For OTA analysis, an elution described elsewhere [63 (link)] was used. The mobile phase flow rate was fixed at 0.3 mL min−1, and the column temperature at 40 °C. MS/MS analysis was performed in MRM (multiple reaction monitoring) mode. The specific MS/MS parameters (retention time, ionization mode, and monitored transitions) associated with specific mycotoxins are shown in Table 6 . The LOD and LOQ for the single aFs were 0.6 and 2.0 µg kg−1, respectively, while for OTA they were 1.5 and 5.0 µg kg−1, respectively.
6
Quantification of Tranexamic Acid in Serum
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TXA concentrations in serum samples were quantified by liquid chromatography-mass spectrometry (LC-MS). Individual, non-pooled, specimens were prepared by adding 200 μL serum to 200 μL of 2.5% perchloric acid and 3 μL of 0.5 μg/mL cis-4 aminocyclohexanecarboxylic acid as the internal standard. These were then vortexed and centrifuged for 10 min at 14,000 rpm. The supernatant was added to LC-MS sample vials containing 150 μL of 0.1 M sodium hydroxide and mixed. Calibration standards and controls were prepared by spiking blank pig serum with known amounts of TXA. The LC-MS system-a 1,290 Infinity HPLC coupled to a 6,550 ifunnel Q-TOF mass spectrometer (Agilent Technologies Inc, Santa Clara, Calif)-was injected with 0.04 μL of sample. The mobile phase consisted of 99% formic acid in water (0.1% w/w-solvent A) and 1% methanol (solvent B). Separation was achieved using an Agilent Technologies ZORBAX Eclipse Plus C18 Rapid Resolution HD column (2.1 mm × 50 mm × 1.8 μm) maintained at 28°C. The mass spectrometer was operated in the positive ion mode and monitored at m/z, amu 158.12 for TXA and 144.10 for the internal standard.
7
Targeted MRM-MS Proteome Quantification
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MRM-MS was performed using an Agilent 6490/6495 series Triple Quadrupole coupled to an Agilent 1290 Infinity UHPLC system (G4220A). Twenty micrograms of each digest were injected onto an Agilent Zorbax Eclipse Plus C18 Rapid Resolution HD column (2.1 × 150 mm, 1.8 μm particles) maintained at 50 °C. The solvents used for the HPLC gradient were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and the following gradient was used at a flow rate of 0.4 mL/min: (%B, time in min): 2, 0; 7, 2; 30, 50; 45, 53; 80, 53.5; 80, 55.5; 2, 56 with a 4 min post-gradient equilibration at 2% B. Transitions were monitored in positive ion mode using dynamic MRM acquisition with a detection window of 1 min, < 900 ms cycle time, and a dwell time of at least 9 ms. All MRM raw data were processed and inspected using the Skyline Daily software.
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